Spliced leader (SL) RNA trans-splicing generates the mature 5' ends of mRNAs by addition of a spliced leader sequence to the 5' end of a pre-mRNA. Trans-splicing is an essential mechanism of gene expression in nematodes. A unique aspect of metazoan trans-splicing is that addition of the spliced leader sequence also brings a new and atypical cap to the mRNA, a trimethylguanosine cap (m2,2,7GpppN) compared to the typical m7GpppN eukaryotic cap. Two populations of mRNAs co-exist in nematodes: 1) non-trans-spliced with a typical m7GpppN cap and variable 5' end sequence and 2) trans- spliced with an m2,2,7GpppN cap and a common 5' 22 nt spliced leader sequence. Mammalian mRNAs only acquire an m7G-cap. Cellular cap-interacting proteins mediate the metabolism of trans-spliced mRNAs and are essential for translation and, therefore, nematode gene expression. The translation initiation factor eIF4E directly binds the mRNA cap. This is the critical and rate limiting step in recruitment of most mRNAs to the ribosome and is a major target for translational control. How nematode eIF4E has adapted to accommodate translation of these two RNA populations remains an important, unanswered question. Nematodes infect almost half the people on earth (~3 billion people) and Ascaris infects ~1 billion people. As mRNA translation in nematodes must differ from the mammalian host, translation of trans- spliced mRNAs provides an attractive target for drug development. We have identified key features of the mechanism of translation of Ascaris mRNAs: 1) Ascaris eIF4E isoforms initiate translation of both trans- spliced and non-trans-spliced nematode mRNAs, yet exhibit a much lower affinity for the m2,2,7G- compared to the m7G-cap; 2) Translation of mRNAs with a m2,2,7G-cap requires a stem loop and specific sequences within the SL that are necessary and sufficient for efficient translation of m2,2,7G-capped mRNAs (the SL effect); 3) Efficient translation of the m2,2,7G-SL mRNAs requires adaptations in Ascaris eIF4E-3 and eIF4G translation initiation proteins; 4) We determined the crystal structures of Ascaris eIF4E-3 bound to the two different caps and defined NMR conformational changes in eIF4E on binding the two caps and SL; and 5) Ascaris has several eIF4E isoforms that translate both types of mRNAs and we hypothesize they translate distinct subsets of mRNAs. Our studies now enable us to mechanistically characterize the SL Effect and nematode translation. We aim to understand how the SL sequence facilitates translation of m2,2,7G-capped mRNAs. We will pursue this goal by 1) determining how Ascaris eIF4E interacts with the m2,2,7G-SL, 2) determining the mechanism(s) through which the SL facilitates translation of m2,2,7G-capped mRNAs, 3) determining the structure of the nematode m2,2,7G-SL and m2,2,7G-SL-eIF4E complex, and 4) determine the role of Ascaris eIF4E isoforms in the translation of different Ascaris mRNAs. These analyses promise to provide important insights into mechanisms of nematode gene expression and adaptation of the translation machinery to trans-splicing in an important group of parasites considered to be Great Neglected Diseases. At the conclusion of these studies, we expect to have a better understanding of how Ascaris eIF4E translates m2,2,7G -capped mRNAs, how Ascaris eIF4E interacts with the trans-spliced SL stem-loop, the structure of the m2,2,7G -SL and m2,2,7G - SL-eIF4E complex, and the potential role of proteins that function in mRNA translation in an important human parasite. Moreover, our studies will provide general insight into translation initiation, eIF4E isoforms, and the role of the 5' UTR element in mRNA translation that will have broad implications for translation in other eukaryotes. PUBLIC HEALTH RELEVANCE: Parasitic nematodes remain a significant public health problem in many parts of the world. Ascaris alone infects upwards of 1 billion people and hinders socioeconomic development in endemic areas. We will carry out studies on novel cap-interacting proteins in Ascaris that are potential targets for new and novel therapeutics against parasitic helminths.